Multiplex Immunofluorescence Staining Protocol

As previously described, mIF staining was conducted using a PANO 7-plex IHC kit (Panovue, Beijing, China) (31 (link)). Antibodies used included anti-panCK (CST4545, Cell Signaling Technology, Danvers, MA, USA), anti-CD8 (CST70306), anti-CD56 (CST3576), anti-CD68 (BX50031, Biolynx, China), and anti-HLA-DR (ab92511, Abcam, UK). The stained slides were scanned, and a single stacked image was constructed using the Mantra System (PerkinElmer, Waltham, MA, US). Furthermore, images of the sections were reconstructed based on a spectral library of multispectral unmixing. Finally, various cells were counted using the InForm image analysis software (PerkinElmer).